ABSTRACT Thymocyte proliferation, survival, and differentiation are tightly controlled by signaling from the pre-TCR. In this study, we show for the first time that the Abelson (Abl) kinases regulate proximal signaling downstream of the pre-TCR. Conditional deletion of Abl kinases in thymocytes reveals a cell-autonomous role for these proteins in T cell development. The conditional knockout mice have reduced numbers of thymocytes, exhibit an increase in the percentage of the CD4(-)CD8(-) double-negative population, and are partially blocked in the transition to the CD4(+)CD8(+) double-positive stage. Moreover, the total number of T cells is greatly reduced in the Abl mutant mice, and the null T cells exhibit impaired TCR-induced signaling, proliferation, and cytokine production. Notably, Abl mutant mice are compromised in their ability to produce IFN-positive CD8 T cells and exhibit impaired CD8(+) T cell expansion in vivo upon Listeria monocytogenes infection. Furthermore, Ab production in response to T cell-dependent Ag is severely impaired in the Abl mutant mice. Together these findings reveal cell-autonomous roles for the Abl family kinases in both T cell development and mature T cell function, and show that loss of these kinases specifically in T cells results in compromised immunity.

[Show abstract][Hide abstract]ABSTRACT:
The development of B cells is dependent on the sequential DNA rearrangement of immunoglobulin loci that encode subunits of the B cell receptor. The pathway navigates a crucial checkpoint that ensures expression of a signalling-competent immunoglobulin heavy chain before commitment to rearrangement and expression of an immunoglobulin light chain. The checkpoint segregates proliferation of pre-B cells from immunoglobulin light chain recombination and their differentiation into B cells. Recent advances have revealed the molecular circuitry that controls two rival signalling systems, namely the interleukin-7 (IL-7) receptor and the pre-B cell receptor, to ensure that proliferation and immunoglobulin recombination are mutually exclusive, thereby maintaining genomic integrity during B cell development.

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The Abelson (ABL) family of nonreceptor tyrosine kinases, ABL1 and ABL2, transduces diverse extracellular signals to protein networks that control proliferation, survival, migration and invasion. ABL1 was first identified as an oncogene required for the development of leukaemias initiated by retroviruses or chromosome translocations. The demonstration that small-molecule ABL kinase inhibitors could effectively treat chronic myeloid leukaemia opened the door to the era of targeted cancer therapies. Recent reports have uncovered roles for ABL kinases in solid tumours. Enhanced ABL expression and activation in some solid tumours, together with altered cell polarity, invasion or growth induced by activated ABL kinases, suggest that drugs targeting these kinases may be useful for treating selected solid tumours.

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The dysregulation of receptor tyrosine kinases (RTKs) in multiple cell types during chronic inflammation is indicative of their pathogenic role in autoimmune diseases. Among the many RTKs, vascular endothelial growth factor receptor (VEGFR) stands out for its multiple effects on immunity, vascularization, and cell migration. Herein, we examined whether VEGFR participated in the pathogenesis of type 1 diabetes (T1D) in nonobese diabetic (NOD) mice. We found that RTK inhibitors (RTKIs) and VEGF or VEGFR-2 antibodies reversed diabetes when administered at the onset of hyperglycemia. Increased VEGF expression promoted islet vascular remodeling in NOD mice, and inhibition of VEGFR activity with RTKIs abrogated the increase in islet vascularity, impairing T-cell migration into the islet and improving glucose control. Metabolic studies confirmed that RTKIs worked by preserving islet function as treated mice had improved glucose tolerance without affecting insulin sensitivity. Finally, examination of human pancreata from patients with T1D revealed that VEGFR-2 was confined to the islet vascularity, which was increased in inflamed islets. Collectively, this work reveals a previously unappreciated role for VEGFR-2 signaling in the pathogenesis of T1D by controlling T-cell accessibility to the pancreatic islets and highlights a novel application of VEGFR-2 antagonists for the therapeutic treatment of T1D.

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2-??) and reprobed with the following Abs for total protein: anti-ZAP70,anti-LAT, and anti-p38 (Cell Signaling Technology); anti-PLC?, anti-ERK1, anti-JNK1, and anti-I?B? from Santa Cruz Biotechnology; anti-Shcfrom BD Transduction Laboratories; and anti-p56lckfrom BioSourceInternational.Immunization and serum Ig measurementsWild-type and Abl/Arg-conditional null mice were immunized i.p. at day0 with 50 ?g of alum-precipitated nitrophenylacetyl (NP)-chicken ?-glob-ulin (T cell-dependent Ag) or NP-LPS (T cell-independent Ag) (BiosearchTechnologies). For T cell-dependent Ag response, mice were challengedwith same dose at day 21. Serum was collected from tail vein at indicatedtimes, and NP-specific IgM and IgG titers were determined by ELISA on96-well plates coated with NP-BSA (Biosearch Technologies), followed byalkaline phosphatase-conjugated goat anti-mouse IgM or IgG, and devel-oped in pNPP substrate (Southern Biotechnology Associates). NP-specificmouse IgM and IgG (gifts from W. Zhang, Duke University Medical Cen-ter, Durham, NC) were used as standards.Generation of Ag-specific T cells afterL. monocytogenes infectionA recombinant L. monocytogenes strain engineered to secrete chickenOVA and pMHC/peptide tetramers was originally provided by M.J. Bevan(University of Washington, Seattle, WA). Wild-type and Abl/Arg-condi-tional null mice were infected i.v. with 5 ? 103CFU of bacteria. Spleno-cytes were prepared at day 7 after infection and were then challenged withor without 0.2 ?M of either OVA (257–264) peptide for CD8 or listerio-lysin O (190–201) peptide for CD4 in the presence of monensin (3 ?M) at37°C for 5.5 h. Cells were surface stained with PE-conjugated anti-CD8 oranti-CD4 Abs, fixed in 2% paraformaldehyde, and permeabilized with0.1% saponin, followed by staining with FITC anti-IFN-? (BD Pharmin-gen) Ab. Activation of Ag-specific CD4 cells was also analyzed by stainingfor both CD4 and CD154. For H-2Kb-OVA-binding experiment, DimerXI (BD Biosciences) was loaded with OVA peptides (257–264) overnight at37°C and preincubated with PE-conjugated anti-mouse IgG1 for 4 h beforeincubation with splenocytes harvested 7 days after L. monocytogenes in-fection. The number of cells binding to the H-2Kbpeptide was analyzed bygating on the CD8?cells. A total of 200,000 cells was collected onFACScan and analyzed by CellQuest.Statistics analysisAll statistics were performed using Student’s unpaired, two-tailed t test.ResultsGeneration of Abl/Arg T cell-conditional knockout miceThe Ablfloxmice were generated, as previously described (20), andwere crossed into the arg?/?background to generate abl flox/floxarg?/?mice. These mice were subsequently crossed with the Lck-Cre transgenic mice to generate conditional loss of Abl kinasesin the T cell lineage. Protein analysis of Abl/Arg-double-nullthymocytes and purified splenic T cells demonstrated the ab-sence of full-length Abl proteins by immunoblotting with an Abspecific for the kinase domain of Abl (Fig. 1, A, upper panel,and B, lane 2). In contrast, Abl protein was present in cells otherthan T cells, indicating specific inactivation of the abl1 gene inT cells (Fig. 1B). Consistent with previous findings (20), a smallamount of a truncated Abl protein was detected in Abl/Arg-nullthymocytes by blotting with Ab specific for the Abl C-terminalregion (Fig. 1A, bottom panel). This truncated version of theAbl protein was previously demonstrated to completely lackAbl kinase activity (20).FIGURE 2.development in Abl/Arg-conditionalknockoutmice.A,Absolutenumbersoftotal thymocytes (wild type (WT), n ?7, 166.7 ? 10.4; Abl/Arg null, n ? 12,73.5 ? 9.5, mean ? SEM). B, Thymo-cytes were stained with anti-CD4 andanti-CD8 Abs, followed by flow cy-tometry analysis. This result is repre-sentative of at least five independentexperiments. C, Absolute numbers ofthe DP cells (WT, 137.1 ? 10.3; Abl/Arg null, 58.7 ? 8.3, mean ? SEM),DN cells (WT, 4.3 ? 0.5; Abl/Arg null,3.8 ? 0.5, mean ? SEM), CD4-SPcells (WT, 18.8 ? 1.6; Abl/Arg null,8.4 ? 1.1, mean ? SEM), and CD8-SPcells (WT, 6.2 ? 0.3; Abl/Arg null,2.7 ? 0.4, mean ? SEM). Horizontalbars indicate mean values. Values of pare indicated.ImpairedthymocyteFIGURE 3.stage in the absence of Abl and Arg kinases. A, Thymocytes were stainedwith anti-CD4, anti-CD8, anti-CD44, and anti-CD25, and flow analysiswas gated on the CD4?CD8?DN population and analyzed for CD25/CD44 expression. The data are representative of at least five independentexperiments. B, Mice were injected i.p. with 20 ?g of anti-CD3? Ab, andthymocytes were stained and analyzed as in A. The data are representativeof three independent experiments.Development of thymocytes is partially blocked at the DN37336Abl KINASES IN T CELL DEVELOPMENT AND FUNCTION

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Thymocyte development is abnormal in Abl/Arg-conditionalknockout miceWe observed that Abl/Arg-conditional knockout mice consistentlyexhibited greater than 50% reduction in the total number of thy-mocytes compared with wild-type controls (Fig. 2A). Thymocytesisolated from the Abl/Arg-conditional knockout mice and theirwild-type littermate controls (4–6 wk old) were surface stainedwith anti-CD4 and anti-CD8 Abs and analyzed by flow cytometry.Although the percentage of CD4?CD8?DP and CD4?or CD8?SP cells was not significantly different between wild-type and mu-tant mice, the percentage of the CD4?CD8?DN population wasconsistently increased by 2- to 4-fold in the Abl/Arg-null mice(Fig. 2B). Comparison of the absolute cell numbers for each sub-population revealed that the reduction in the total number of thy-mocytes in the Abl/Arg mutant mice compared with wild-typemice was primarily due to a ?50% decrease in DP cells as well asFIGURE 4.TCR signaling (A–E). SL-12?12 cells were pretreatedwith or without the Abl kinase inhibitor imatinib (5?M) for 1 h. Cells were then activated with biotinylatedanti-TCR? plus streptavidin for the indicated times at37°C. Equal amount of protein lysates was loaded ontoSDS-PAGE and analyzed by Western blotting with Absspecific for phosphorylated (p) ZAP70/Syk (A), LAT(B), PLC?1 (C), Shc (D), and ERK (E). Membraneswere stripped and reprobed with Abs to each of the cor-responding total proteins.Abl kinase activity is required for pre-FIGURE 5.duced in the Abl/Arg-conditionalknockout mice. A, Splenocytes iso-lated from 5- to 9-wk-old mice werestained with anti-TCR?, CD4, CD8,and B220 Abs, followed by flow cy-tometry analysis. Percentage of posi-tive cells in the gated region is indi-cated. B, Absolute numbers of totalsplenocytes (n ? 10, WT, 102.6 ?10.8; Abl/Arg null, 93.9 ? 9.8,mean ? SEM), total T cells (n ? 8,WT, 43.3 ? 4.4; Abl/Arg null,28.5 ? 3.5, mean ? SEM), CD4?cells (n ? 8, WT, 26.5 ? 2.3; Abl/Arg null, 17 ? 1.6, mean ? SEM),and CD8?cells (n ? 10, WT, 11.2 ?1.1; Abl/Arg null, 6.6 ? 0.8, mean ?SEM). Horizontal bars indicate meanvalues. Values of p are indicated.Splenic T cells are re-7337 The Journal of Immunology

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similar decreases in CD4-SP and CD8-SP cells, with no significantdifference in the total number of DN cells (Fig. 2C). These resultssuggested potential developmental defects during the DN to DPtransition in the absence of Abl and Arg kinases.The DN thymocytes can be subdivided into four developmentalstages (DN1, DN2, DN3, and DN4) based on the expression of theCD25 and CD44 cell surface molecules (2, 3). These stagesprogress in thefollowing order:CD25?CD44?(DN2), CD25?CD44?(DN3), and CD25?CD44?(DN4). TCR? gene rearrangements are initiated at the DN2 stageand continue during the DN3 stage. The rearranged TCR? chainheterodimerizes with the pre-T? chain to form the pre-TCR at thecell surface. Signals derived from the pre-TCR at the DN3 stageare important for maturation to the DN4 stage and are implicatedin proliferative expansion, survival, allelic exclusion of the TCR?locus, and induction of TCR? rearrangement (2). To define thedefect in thymocyte development in the Abl/Arg-conditionalknockout mice, we analyzed the expression of CD25 and CD44among the DN populations. Thymocytes from Abl/Arg-condi-tional knockout mice consistently presented a higher percentage ofCD25?CD44?(DN1),CD25?CD44?(DN3) cells with a corresponding decrease in DN4stage cells compared with wild-type mice (Fig. 3A), which sug-gests that loss of Abl kinases may affect the DN3 to DN4 transitionduring thymocyte development. Successful TCR? gene rearrange-ment and expression of the pre-TCR occur at DN3 stage, and func-tional signaling through pre-TCR is critical for thymocyte devel-opment (2). Therefore, we analyzed the expression of TCR? onsubsets of thymocytes by FACS analysis. There was no significantdifference in surface expression of TCR? in all four thymocytesubpopulations between wild-type and Abl/Arg mutant mice (datanot shown). Thus, Abl kinases are not involved in TCR? chainrearrangement and surface expression. Similarly, analysis of pos-itive selection by staining thymocytes for DP expression of TCR?and CD69 (21) demonstrated that there was no significant differ-ence between Abl/Arg-null thymocytes and their littermate con-trols (data not shown).To address whether the defect in the transition from DN3 toDN4 elicited by the absence of Abl kinases could result from im-paired signaling downstream of the pre-TCR, we performed invivo injections with anti-CD3? Ab, which activates the pre-TCRFIGURE 6.wild-type, c-Abl-null, Arg-null, and Abl/Arg-double-null mice were stimulated with either anti-CD3 alone (top panel) or anti-CD3 plus anti-CD28 (bottompanel) Abs at the indicated concentrations for 48 h, and [3H]thymidine was added during the last 16–20 h of culture. Data are mean ? SD of triplicate wells.Thisexperimentisrepresentativeofatleastthreeindependentexperiments.B,Splenocytesisolatedfromwild-typeorAbl/Arg-conditionalnullmicewereincubatedwith allogeneic stimulators (MLR) for 72 h. Proliferation was measured by [3H]thymidine incorporation in triplicate wells (mean ? SD). This experimentis representative of at least five independent experiments. C, Wild-type or Abl/Arg-double-null T cells were stimulated with either anti-CD3 (5 ?g/ml)alone, or anti-CD3 (1 ?g/ml) plus anti-CD28 (5 ?g/ml) Abs, as indicated. Culture supernatant was collected after 24 h, and the levels of IL-2 (top panel),IL-4 (middle panel), and IFN-? (bottom panel) were measured by ELISA in triplicates. This result is representative of four independent experiments. Thepercentage of decrease in cytokine levels in mutant T cells compared with wild-type T cells stimulated with anti-CD3 or anti-CD3 plus CD28 was calculatedas follows. For IL-2: 72.8% ? 10.7 (CD3); 72.9% ? 5.6 (CD3 ? CD28). For IL-4: 40.1% ? 32.3 (CD3); 31.6% ? 28.5 (CD3 ? CD28). For IFN-?: 63.7%? 21.6 (CD3); 74.3% ? 17.3 (CD3 ? CD28). D, Abl protein was absent in Cre-GFP cells after in vitro retroviral-Cre infection (left panel). Vector-GFP(wild-type) and Cre-GFP (Abl/Arg-null) T cells were stimulated with anti-CD3 Ab before retroviral infection and analyzed for proliferation in the presenceof IL-2 for 24 h (middle panel). Supernatant was collected after restimulation of indicated cells with anti-CD3 for 6 h, and IFN-? levels were measured(right panel). The data are representative of three independent experiments and retroviral infections.Proliferation and IL-2 secretion are impaired in mice lacking Abl and Arg in the T cell compartment. A, Splenic T cells isolated from7338Abl KINASES IN T CELL DEVELOPMENT AND FUNCTION

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and accelerates thymocyte maturation (22, 23). Mice were injectedi.p. with anti-CD3? Ab and thymocytes were analyzed 4 days later.As expected, wild-type mice exhibited accelerated maturationthrough the DN3 stage with few cells remaining at the DN2 andDN3 stages (Fig. 3B). In contrast, a high number of DN3 cellsaccumulated in the Abl/Arg-double-null thymocytes (Fig. 3B). To-gether our data support a role for Abl kinases in thymocyte devel-opment and pre-TCR signaling in vivo.Inhibition of Abl kinases impaired pre-TCR signalingTo directly examine the role of Abl kinases in pre-TCR signaling,we used the pre-T cell line SL-12?12, derived from a spontaneousSCID mouse thymoma that stably expresses functionally rear-ranged TCR? with endogenous pre-TCR? chains on the cell sur-face (24). To assess whether loss of Abl kinase activity affectedpre-TCR signaling, SL-12?12 cells were pretreated with the Ablkinase inhibitor imatinib mesylate (5 ?M for 1 h), followed bystimulation with biotinylated anti-TCR? Ab and cross-linking withstreptavidin for the indicated times (Fig. 4). We observed en-hanced phosphorylation of ZAP70/Syk, LAT, and PLC?1, as wellas the adaptor protein Shc following pre-TCR activation in controlcells. In contrast, the tyrosine phosphorylation of these moleculeswas greatly diminished in cells treated with the Abl kinase inhib-itor (Fig. 4, A–D). Phosphorylation of ERK was also strongly en-hanced after pre-TCR cross-linking in control cells, and it was onlyslightly reduced at the earliest time point after pre-TCR stimula-tion in the imatinib-treated cells (Fig. 4E). Together these resultssuggest that Abl kinases are involved in pre-TCR signaling.Decreased proliferation and cytokine production ofAbl/Arg-double-null mature T cellsSplenocytes were isolated from Abl/Arg-conditional knockoutmice and their littermate controls (5–9 wk), and stained with cellsurface markers for T and B cells, including TCR?, CD4, CD8,and B220. We observed a ?40–50% decrease in the percentage oftotal T cells, as well as CD4?and CD8?cell populations, and arelative small increase in the percentage of B cells in the Abl/Arg-conditional null compared with wild-type mice (Fig. 5A). B cellproliferation was not affected in these mice because there was nosignificant difference in proliferation following stimulation of totalsplenocytes with the B cell mitogens anti-IgM or LPS (data notshown). Although the absolute number of total splenocytes wasnot significantly different between mutant and wild-type mice, thetotal T cell numbers were significantly reduced in the absence ofAbl family kinases, with significant reductions in both CD4?andCD8?subsets in the Abl/Arg-conditional knockout mice (Fig. 5B).The reduction in splenic T cells in the mutant mice suggested thatAbl kinases might be involved in T cell proliferation or survival.Splenic T cells from wild-type, Abl-null, Arg-null, and Abl/Arg-double-null mice were stimulated with either anti-CD3 alone oranti-CD3 plus anti-CD28 for 48 h, and proliferation was measuredby [3H]thymidine incorporation (Fig. 6A). Resting T cells did notshow significant [3H]thymidine incorporation in both wild-typeand mutant cells (data not shown). Abl- and Arg-single-null T cellsshowed a significant reduction in proliferation upon TCR stimu-lation with low amounts (1 ?g/ml) of anti-CD3 (Fig. 6A). Athigher doses of anti-CD3 (5 ?g/ml) alone or anti-CD3 plus higherdose of anti-CD28 (5 ?g/ml), Arg-null T cells proliferated similarto wild-type controls. Notably, loss of both Abl and Arg resultedin a more profound defect in T cell proliferation compared with theloss of either kinase alone at both low and high doses of anti-CD3in the absence or presence of CD28 costimulation (Fig. 6A). Additionof IL-2 failed to reverse the proliferation defect in the Abl/Arg-dou-ble-null T cells (data not show). We did not observe significant dif-ferences in proliferation between wild-type and Abl/Arg-double-nullT cells in response to PMA and ionomycin stimulation (data notshown). We also observed significantly less proliferation in lympho-cytes from Abl/Arg-conditional null mice compared with wild-typemice in response to allogeneic stimulation in a MLR (Fig. 6B). Thus,loss of Abl/Arg kinases does not result in a general defect in T cellproliferation, but specifically inhibits TCR-induced cell proliferation,and suggests that Abl kinases function to transduce proximal signalsdownstream of the TCR.To study whether T cell function is impaired in the absence ofAbl kinases, wild-type or Abl/Arg-double-null T cells were stim-ulated with anti-CD3 in the absence or presence of anti-CD28 Absfor 24 h, and the levels of IL- 2, IL-4, and IFN-? secreted intoculture supernatant were measured by ELISA (Fig. 6C). Both IL-2and IFN-? levels were markedly reduced by ?70% in Abl/Arg-double-null T cells compared with wild type (Fig. 6C, top andbottom panels), whereas a smaller reduction of IL-4 (?30%) wasobserved in the Abl/Arg mutant mice (Fig. 6C, middle panel).Thus, Abl kinases regulate Th function, and specifically TCR-me-diated IL-2 and IFN-? secretion.To determine whether the defect in mature T lymphocyte pro-liferation and cytokine secretion in the absence of Abl kinases isdue to defective T cell development or to an intrinsic cell-auto-nomous defect, we have deleted the abl gene in vitro followingtransduction with a retroviral Cre vector. T cells were stimulatedwith anti-CD3 before retroviral infection, and deletion of the ablFIGURE 7.Wild-type or Abl/Arg-double-null T cells were stimulated with anti-CD3for 48 h, and BrdU was added during the last 30 min of culture. Fortreatment with the Abl kinase inhibitor, imatinib (5 ?M) was added towild-type T cells. Cells were stained with anti-BrdU Ab and PI, and ana-lyzed by flow cytometry. Percentage of S-phase cells is indicated. B, Wild-type or Abl/Arg-null T cells were stimulated with plate-bound anti-CD3 (5?g/ml) for 48 h. Cells were stained with annexin V and 7-aminoactino-mycin D, followed by flow analysis. C, Wild-type or Abl/Arg-null T cellswere stimulated with plate-bound anti-CD3 (5 ?g/ml) for the indicatedtimes. Cell lysates were separated on SDS-PAGE and analyzed using eitheranti-caspase 3 or anti-cleaved caspase 3 Abs, as indicated.Decreased cell cycle entry in the absence of Abl kinases. A,7339The Journal of Immunology

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gene was confirmed by the absence of Abl protein (Fig. 6D, left).Proliferation of the CD3-stimulated T cells was measured in thepresence of IL-2 for 24 h, and IFN-? secretion was measured 3–8h after restimulation with anti-CD3 Ab. We observed a significantreduction in both proliferation and IFN-? secretion in the Abl/Arg-double-null T cells compared with wild-type T cells (Fig. 6D, mid-dle and right panels). Thus, Abl kinases have a T cell-autonomousrole and are required for maximal proliferation and cytokine se-cretion in mature T cells. Prolonged restimulation of these acti-vated T cells with anti-CD3 Ab resulted in apoptosis of both wild-type and Abl/Arg-null cells (data not shown).To further investigate the possible mechanism(s) responsible forthe reduced T cell proliferation induced by loss of the Abl kinases,cells were pulse labeled for a short time with BrdU at the end ofanti-CD3 stimulation, and the cell cycle was analyzed by the DNAprofile. Abl/Arg-double-null T cells or wild-type T cells treatedwith the Abl kinase inhibitor imatinib showed a consistently lowerpercentage of cells entering into S phase compared with wild-typeT cells (Fig. 7A). There was no significant difference in the per-centage of sub-G1cells, suggesting no increased apoptosis in Abl/Arg-double-null T cells. Moreover, analysis of T cell survivalupon TCR stimulation using annexin V and PI staining (Fig. 7B),as well as Western blot analysis of caspase 3 activation (Fig. 7C)did not show differences in apoptosis between wild-type and Abl/Arg-double-null T cells. These results indicate that the reduced Tcell proliferation in the absence of Abl kinases is primarily due todecreased cell cycle entry.Abl kinases are involved in multiple TCR signaling pathwaysTo define the possible mechanism(s) responsible for impaired Tcell proliferation, we examined expression of early T cell surfacemarkers following TCR activation. Wild-type and Abl/Arg-dou-ble-null T cells were stimulated with anti-CD3 Ab for 18 h andstained with either anti-CD25? (IL-2R? chain) or the CD69 earlyT cell activation marker. There was no significant difference in theinduction of either CD25? or CD69 expression between wild-typeand mutant T cells (data not shown), indicating that the prolifer-ation defect in the Abl/Arg-double-null T cells is not due to de-creased IL-2R on the cell surface. These findings suggested thatinhibition of cell proliferation in T cells lacking Abl and Arg ki-nases may result from impaired TCR-mediated signaling. Indeed,we found that TCR-induced phosphorylation of ZAP70, PLC?1,and LAT was greatly reduced in Abl/Arg-double-null T cells com-pared with wild-type cells (Fig. 8, A–C). These results are consis-tent with our previous findings of reduced phosphorylation of thesesignaling proteins in imatinib-treated primary T cells and splenic Tcells derived from abl1?/?abl2?/?mice (14). Notably, TCR-in-duced Lck activation measured by tyrosine 394 phosphorylationwas unchanged in T cells lacking Abl family kinases comparedwith wild-type controls (data not shown). We also analyzed thephosphorylation of the Shc adaptor protein following TCR stim-ulation of purified T cells derived from control and Abl/Arg-con-ditional knockout mice, and show for the first time that Abl/Arg-null T cells exhibit a dramatic decrease in Shc tyrosinephosphorylation following TCR stimulation (Fig. 8D). ActivationoftheMAPKpathwaysfollowingTCRstimulationwasalsoimpairedin Abl/Arg-double-null T cells, with loss of the Abl kinases leading toamoreseverereductioninJNKactivationthanthatobservedforERKactivation (Fig. 8, E and F). In contrast, we found that phosphoryla-tion of the p38 MAPK was unchanged in Abl/Arg-double-null T cellscompared with wild-type T cells (Fig. 8G).We have shown in this study that IL-2 production was markedlyreduced in Abl/Arg-null T cells, and have previously observed thatinhibition of Abl kinase activity reduced activation of theFIGURE 8.indicated. Equal amounts of lysates were analyzed by Western blotting with Abs specific for phosphorylated (p) ZAP70 (A), LAT (C), PLC?1 (B), Shc (D),JNK (E), ERK (F), p38 (G), and I?B? (H). Membranes were stripped and reprobed with Abs to measure total levels of the corresponding proteins.TCR signaling is impaired in Abl/Arg-double-null mature T cells (A–H). Wild-type and Abl/Arg-double-null T cells were stimulated, as7340 Abl KINASES IN T CELL DEVELOPMENT AND FUNCTION

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CD28RE/AP response element of the IL-2 promoter followingTCR activation in Jurkat T cells (14). The NF-?B transcriptionfactor is known to bind to the CD28RE region and activate IL-2gene transcription (25), and is also required for chromatin remod-eling of the IL-2 promoter upon TCR stimulation (26). Thus, weexamined whether NF-?B activation was impaired in T cells lack-ing the Abl kinases. Inactive NF-?B is sequestered by binding toI?B in the cytoplasm of resting T cells. Upon TCR activation,I?B? is phosphorylated, which triggers the ubiquitination and deg-radation of I?B?, thereby releasing NF-?B and promoting itstranslocation to nucleus to regulate transcription (27, 28). Asshown in Fig. 8H, phosphorylation of I?B? was markedly de-creased in Abl/Arg-double-null T cells compared with wild-typecontrols following stimulation with either anti-CD3 alone or anti-CD3 plus anti-CD28. Thus, decreased IL-2 production in the ab-sence of Abl kinases may be mediated in part by decreased NF-?Bactivation.Abl kinases are required for T cell function in vivoTo determine whether Abl kinases play a role in T cell function invivo, mice were injected with T cell-dependent or T cell-indepen-dent Ags, and Ab production was analyzed in Abl/Arg-conditionalknockout mice and control littermates. As shown in Fig. 9A, wild-type mice produced high levels of IgM in response to initial in-jection of T cell-dependent Ag, and high levels of IgG upon sec-ondary boost with same Ag. In contrast, Abl/Arg-conditionalknockout mice were markedly impaired in both early IgM secre-tion and secondary isotype switch to IgG secretion, suggesting thatTh function is severely impaired in these mice. As expected,wild-type and mutant mice secreted equal amounts of IgM inresponse to a T cell-independent Ag (Fig. 9B), indicating that Bcell function is not affected in the Abl/Arg T cell-conditionalknockout mice.FIGURE 9.conditional null mice. Wild-type and Abl/Arg-conditional null micewere injected i.p. with either T cell-dependent Ag (NP-CGG) (A) or Tcell-independent Ag (NP-LPS) (B). Serum was collected from the tailvein at the indicated time points. Levels of IgM and IgG were measuredby ELISA; data are mean ? SD of triplicate wells. This result is rep-resentative of four (T cell-independent Ag) and five (T cell-dependentAg) experiments.T cell-dependent Ab secretion is reduced in Abl/Arg-FIGURE 10.with L. monocytogenes through tail vein injection. After 7 days, splenocytes were cultured with or without specific OVA (A) or listeriolysin O (D)peptides for 5.5 h. Cells were then surface stained with anti-CD8 (A) or anti-CD4 (D), respectively, and intracellular stained with anti-IFN-? Abs,followed by flow cytometry analysis. B and E, Quantitation of absolute numbers of CD8?IFN-??(B) or CD4?IFN-??(E) DP cells from wild-typevs Abl/Arg-conditional null mice. For IFN-??CD8?, WT, n ? 9, 1.2 ? 0.5; Abl/Arg null, n ? 8, 0.2 ? 0.1. For IFN-??CD4?, WT, n ? 4, 4.9 ?1.6; Abl/Arg null, n ? 4, 2.2 ? 1.2. (mean ? SD). Horizontal bars indicate mean values. C, Total number of CD8?T cells binding to H-2Kbpeptide.This is a representative experiment of four independent infections with L. monocytogenes (WT, 2.6 ? 0.8; Abl/Arg null, 0.7 ? 0.5, mean ? SD).F, Total number of CD4?/CD154?DP cells. This is a representative experiment of four independent Listeria infections (WT, 1.9 ? 0.6; Abl/Argnull, 0.8 ? 0.3, mean ? SD). Values of p are indicated.Defective production of CTL in Abl/Arg-conditional null mice. A and D, Wild-type and Abl/Arg-conditional null mice were infected7341 The Journal of Immunology

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To investigate whether deficiency in the expression of Abl fam-ily kinases results in altered susceptibility to infection, Abl/Arg-conditional knockout mice and littermate controls were infectedwith a low dose of L. monocytogenes, and the mice were analyzedfor the development of Ag-specific CD8?and CD4?T cells. Wefound that Abl/Arg mutant mice developed very few Ag-inducedIFN-?-producing CD8?effector T cells compared with wild-typemice (Fig. 10A). Notably, the total number of IFN-?-positiveCD8?T cells in the Abl/Arg-conditional knockout mice was re-duced by greater than 80%, and it was only 18% compared withthe wild-type controls (Fig. 10B). The number of specific CD8?Tcells binding to the H-2Kbpeptide was also significantly reducedin the Abl/Arg-null mice (73% reduction compared with the wildtype; Fig. 10C). Thus, expansion of specific CD8?T cells uponListeria infection was impaired in Abl/Arg-null mice. In contrast,the percentage of IFN-?-positive CD4?T cells was similar be-tween wild-type and mutant mice (Fig. 10D). However, the totalnumber of IFN-?-positive CD4?T cells was ?50% of the wildtype (Fig. 10E), which most likely reflects the reduced number ofsplenocytes in the mutant mice after infection (52.6% of the wild-type mice). The total number of CD4?/CD154?DP cells in themutant mice was also ?50% of the wild-type mice (Fig. 10F).Together, these results reveal that Abl kinases are principally re-quired for the development of effector CD8?T cells in vivo inresponse to Listeria infection.DiscussionOur work has uncovered cell-autonomous roles for Abl familykinases in the regulation of both thymocyte development and ma-ture T cell function, and showed that Abl kinases signal down-stream of both the pre-TCR and TCR. The generation of T cell-specific Abl/Arg-null mice permitted us to bypass the embryoniclethality associated with complete loss of Abl and Arg, and re-vealed a previously unappreciated role for these kinases in thymo-cyte development. We found that Abl/Arg-double-null thymocyteswere partially blocked at the DN3 stage, a developmental stagethat requires signaling through the pre-TCR to progress to the DN4stage (2). Abl/Arg-double-null thymocytes expressed wild-typelevels of TCR? on the DN populations, which suggests that theAbl/Arg-double-null thymocytes have normal amounts of pre-TCR surface expression. Rather, we found that signaling throughthe pre-TCR was impaired in the absence of Abl kinases. In thisregard, Abl/Arg-double-null thymocytes failed to progress throughthe DN3 stage induced by in vivo injection of anti-CD3? Ab,which results in complete mobilization of wild-type thymocytes tothe DN4 stage with few remaining DN3 stage cells. Moreover, lossof Abl kinase activity produced a marked decrease in the activationof several signaling molecules in pre-TCR cells.Knockout mouse studies have identified a critical role for theLck and ZAP70/Syk tyrosine kinases in thymocyte development(4, 5). Loss of Lck or ZAP70 and Syk results in arrested thymocytedevelopment with a partial or complete block at the DN3 stage.Similarly, genetic inactivation of adaptor proteins regulated down-stream of these tyrosine kinases, such as LAT (9) and Shc (8),results in arrested thymocyte development. Our finding that Ablkinase activity is required for maximal activation of ZAP70/Syk,LAT, PLC?1, and Shc following pre-TCR activation defines a newrole for Abl family kinases downstream of the pre-TCR inthymocytes.As expected from our finding that CD4?and CD8?SP thymo-cytes were decreased in the Abl/Arg-conditional null mice, maturesplenic T cells were also significantly reduced by ?40–50% in themutant mice. Although Abl- and Arg-single-null T cells showed alimited defect in proliferation, Abl/Arg-double-null T cells exhib-ited a more profound proliferative defect upon anti-CD3 and/oranti-CD28 stimulation. Thus, both Abl and Arg kinases are re-quired for maximal immune responses. The basis for the prolifer-ative defect in T cells lacking Abl kinases is most likely due todelayed cell cycle entry into S phase. A role for Abl kinases in cellcycle progression is consistent with previous findings that Abl isrequired for growth factor-induced DNA synthesis in fibroblasts(29–31). Furthermore, inhibition of TCR-induced proliferation incells lacking Abl and Arg is also consistent with a role for Ablkinases in the regulation of mature TCR signaling. Our currentfindings and previous data (14) suggest that Abl kinases may linkTCR engagement and Lck activation to the activation of ZAP70/Syk, LAT, and PLC?1. Alternatively, Abl kinases may not directlyactivate ZAP70 and specific signaling molecules, but rather Ablkinases may regulate the assembly of signaling complexes and/orcytoskeletal scaffolds required for proper activation of ZAP70 anddownstream signaling targets.We have also shown that Abl kinases are involved in maximalactivation of Shc, ERK, and JNK in mature T cells. FollowingTCR engagement, the adaptor protein Shc is recruited to the TCRthrough interactions with ZAP70 and CD3? (32, 33). Shc proteinshave been reported to be phosphorylated by Lck and Syk/ZAP70tyrosine kinases (34), and subsequently bind to Grb2 and son ofsevenless, leading to activation of the Ras-MAPK pathway (35).We found that phosphorylation of Shc at tyrosines 238/240 fol-lowing TCR stimulation was greatly reduced in Abl/Arg-double-null T cells. Thus, Abl kinases may regulate Shc activity indirectlythrough the regulation of ZAP70/Syk phosphorylation, or may di-rectly phosphorylate Shc at Y238/Y240. Tyrosine phosphorylationof Shc is important for both T cell proliferation and IL-2 produc-tion (35, 36), and Abl-mediated phosphorylation of Shc may playa role in the regulation of these processes. Furthermore, decreasedIL-2 production in Abl/Arg-null T cells is most likely linked todecreased transcriptional activity of factors required for IL-2 geneexpression, such as NF-?B. In this study, we showed that TCR-induced NF-?B activity, as measured by the phosphorylation ofI?B?, is decreased in the absence of the Abl kinases. Additionally,JNK activity is reduced in Abl/Arg-double-null T cells, which maycontribute to the impaired production of cytokines, includingIFN-?, observed in Abl/Arg T cell-conditional knockout mice inresponse to TCR stimulation and bacterial infection. In this regard,JNK activity was shown to be required for production of IFN-? inT cells (37).A most striking finding derived from the analysis of the Abl/ArgT cell-conditional null mice was the discovery that these mice areimpaired in the development of Ag-specific CTL and display amarked reduction of IFN-?-positive CD8?T cells compared withwild-type mice in response to L. monocytogenes infection. More-over, the Abl/Arg-double-null mice fail to mount a response to Tcell-dependent Ags. Thus, Abl kinases are required for both hu-moral and cellular immunity. The findings presented in this studywith the Abl/Arg-conditional knockout mice support the notionthat loss of Abl family kinases specifically in T cells underlies theincreased susceptibility to infection observed in Abl1 knockoutmice (18, 19), as well as the reported immunosuppressive effects ofimatinib treatment (38, 39).L. monocytogenes is a potent inducer of CD8 T cell responses,and once primed to proliferate by the pathogen, CD8 T cells un-dergo proliferation as well as differentiation into effector and mem-ory T cells (40). Recent work has suggested that asymmetric celldivision of CD8 T cells proliferating in response to L. monocyto-genes infection results in the segregation of immune receptors andpolarity proteins following formation of the immunological syn-apse, thereby producing effector CTL derived from the daughter7342 Abl KINASES IN T CELL DEVELOPMENT AND FUNCTION